KR20010012895A - New bpc peptide salts with organo-protective activity, the process for their preparation and their use in therapy - Google Patents

Abstract

The present invention discloses new pharmaceutical compositions useful for the treatment of various human and animal diseases. These pharmaceutical compositions contain one or more salts of BPC (Body Protection Compounds) fragments of 8-15 amino acids or analogs thereof. The cations of the salts are derived from inorganic or organic bases.

Description

Novel body protective compound peptide salts with organ protective activity, methods for their preparation and use in therapies {NEW BPC PEPTIDE SALTS WITH ORGANO-PROTECTIVE ACTIVITY, THE PROCESS FOR THEIR PREPARATION AND THEIR USE IN THERAPY}

WO92 / 04368 discloses BPCs having a molecular weight of about 40,000 and having body protective activity, their preparation and their use. WO93 / 24521 and WO94 / 11394 disclose BPC peptides having the same type of long-term protective activity as known as parent protein BPC. Sikiric et al., Digestive Diseases and Sciences, 41 (1996) 7, 1518-1526, disclose that pentadecapeptide, when dissolved in water and saline, has a beneficial prophylactic effect on acute pancreatitis and subsequent gastroduodenal damage in rats. BPC 157 is described.

Thus, BPC peptides are known to have a wide variety of pharmaceutical uses. However, the physicochemical stability of such peptides is not satisfactory, for example in normal saline. In addition, administration of BPC peptides, particularly by injection of normal saline or aqueous solution, can cause pain and / or necrosis.

Salts of peptides and proteins are well known in the art. For example, Bertrand M. et al., Journal of Peptide Research, 49 (1997) 3, 269-272, describe that certain salts have very high selectivity with respect to the structure and stability of the peptide. For example, addition of a monovalent cation, such as NH ⁺ _4, to a solution of peptide [Glu-Leu] at a final concentration of 0.1 M induces a transition to a water soluble beta structure. In contrast, it was observed that this transition does not occur when using Li ⁺ , Na ⁺ or Cs ⁺ ions.

Of ion pairs with surface accessibility in protein stability by measuring the effect of added salts (KCl, MgCl ₂ and LaCl ₃ ) on the stability of the newly constructed double-stranded alpha-helical coils at neutral and acidic pH The role was investigated. The results show that the added salt can have a complex effect on protein stability, including stabilization contributions and destabilization contributions, so that the overall effect depends on the nature of the interrelationship of charged residues and ions present in the protein. Kohn et al., Journal of Molecular Biology, 267 (1997) 4, 1039-1052.

In addition, experiments with conventional model peptides suggest that salt bridges separated by i, i +4 along the peptide chain are more stable than those spaced by i, i +3, and the base from the N-terminus to the C-terminus- It can be seen that the acid-base order is preferred to acid. However, at present, it is not known whether the surface salt bridge has a strong stabilizing effect on the original structure in the protein. Berger et al., Journal of Biomolecular Structure and Dynamics, 14 (1996) 3, 285-291.

Therefore, the properties of peptide salts in terms of stability, structure and action have a great impact on certain ions and other intrinsic or external factors involved in forming a particular salt. At present, it is not possible to predict what specific properties a particular salt of a particular peptide will have.

The technical problem associated with the present invention is to provide a BPC peptide in a more stable form, and to provide a diagnostic and / or pharmaceutical composition comprising a BPC peptide exhibiting improved stability and at least the same pharmacological activity as the BPC peptide itself. Is that. In addition, the technical problem associated with the present invention is to overcome the above-mentioned disadvantages, and in particular to provide a pharmaceutical composition that allows for injectable painless injection of the BPC peptide.

The present invention provides a novel dosage form of synthetic body protection compound (BPC) peptides comprising 8 to 15 amino acid residues having a molecular weight of 900 to 1,600 daltons having organ protective activity, preparations thereof, diagnosis and treatment To its use in.

Proteins and peptides are known to be useful in treating a variety of diseases in humans and animals. Many such agents are produced in vivo and can be extracted from animals or humans to prepare pharmaceutical compositions. Examples of pharmaceutically useful proteins or peptides include insulin, erythropoietin, BMP, interferon and the like. Other examples include gastric juice proteins that have recently been isolated successfully and have mucosal protective activity designated BPC.

1 shows the IR spectrum of NaBPC157.

2 shows the IR spectrum of Na ₂ BPC157.

3 shows the IR spectrum of the cesium salt of BPC157 (Cs ₂ BPC157).

4 shows the IR spectrum of the TRIS salt of BPC157 (TRIS-BPC157).

5 shows the IR spectrum of the di-TRIS salt of BPC157 [(TRIS) ₂ BPC157].

6 shows the IR spectrum of the 2-aminopropanol salt of BPC157 (2-AMP-BPC157).

7 shows the IR spectrum of the triethanolamine salt of BPC157 (TEAM-BPC157).

The present invention achieves these problems by providing a pharmaceutical or diagnostic composition comprising a BPC peptide salt and a pharmaceutically or diagnostically effective amount of the BPC peptide salt, wherein the anion of the salt has a molecular weight of 900 to 1,600 Daltons A negatively charged peptide comprising 8 to 15 amino acids having the formula (I), wherein the cation of the salt is a cation of a nontoxic and pharmaceutically acceptable base of an inorganic or organic.

[Zaa Pro Pro Pro Xaa Yaa Pro Ala] ^{(-) or (2-)}

In formula (I), Xaa is a neutral aliphatic amino acid residue, in particular Ala, bAla, Leu, Ile, Gly, Val, Nle or Nva.

Yaa is a basic amino acid residue, in particular Lys, Arg, Orn or His.

Zaa is an acidic amino acid residue, in particular Glu, Asp, Aad or Apm.

In particular, the cations of the salts are alkali or alkaline earth metals, such as Na ⁺ , K ⁺ , Li ⁺ , Cs ⁺ , Ca ²⁺ or other metals such as Zn ²⁺ or primary, secondary or tertiary amines or organics. Compounds such as NH ₄ ⁺ , triethanolamine ⁺ , cyclohexylamine ⁺ , 2-AMP ⁺ (2-amino-1-propanol) or TRIS [tris- (hydroxymethyl) aminomethane], but these cations are physiologically acceptable It should be possible.

The BPC peptide salts of the present invention surprisingly have at least the same pharmaceutical activity as BPC peptides and have significantly increased physicochemical stability compared to acetate of free BPC peptides or BPC peptides. The cation used according to the present invention does not affect BPC peptide activity, but increases its stability. The BPC peptide salts of the present invention are much more stable than BPC peptide or BPC peptide acetate, for example in normal saline or water. In addition, the salts of the present invention are suitable for oral administration and do not exhibit any unwanted side effects such as pain or necrosis during or after administration, in particular by injection. Thus, the BPC peptide salts of the present invention have improved efficacy for small intestinal transfusion and parenteral administration. In addition, the salts of the present invention are highly preferred because they do not show any signs of toxicity at doses up to 50 mg / kg body weight.

The salts of the present invention may be prepared by dissolving the free BPC peptide in a suitable solvent or in an other suitable solvent or in an aqueous solvent or an aqueous / alcoholic solvent, and then evaporating, cooling and lyophilizing the solution or other solvents, eg For example, diethyl ether can be obtained by adding to the aqueous solution and / or the alcoholic solution of the BPC peptide salt separating the insoluble crude salt to separate the salt of the present invention obtained above. For salt formation, one mole or up to two moles of base, ie cations and one mole of free BPC peptide, are used. For alkali BPC peptide salt preparation, preference is given to using alkali metal carbonates or alkali metal hydrogen carbonates. The prepared peptide salt can be easily dissolved in water. Therefore, the present invention also relates to a process for the preparation of BPC peptide salts.

In the context of the present invention, the term base is considered to be a substance capable of forming cations in solution, in particular in aqueous and aqueous / alcoholic solutions.

In the context of the present invention, the term pharmacological activity includes prophylactic and therapeutic activity. Thus, pharmaceutical compositions refer to compositions having prophylactic and / or therapeutic activity.

The present invention also relates to pharmaceutical or diagnostic compositions comprising, together with the BPC peptide salts of the invention, optionally one or more pharmaceutically acceptable carriers, as well as methods of making the compositions. Such compositions are suitable for topical or systemic administration and are suitable, for example, for formulations such as injectable solutions, tablets, creams, capsules, ointments, lotions, lingualette and the like. The dosage is preferably in the range of 10 ⁻⁵ to 10 ⁻² mg / kg body weight at a high concentration of 0.1 to 0.5% when administered systemically or topically. Optimal dosages for particular treatments can be determined by one skilled in the art.

The present invention provides pharmaceutical preparations as described above which contain, besides one or more of the BPC peptide salts of the invention, especially trehalose and / or pharmaceutically or diagnostically acceptable carriers, diluents and / or additives for oral administration. It relates to a diagnostic composition.

In addition to the BPC peptide salts, the compositions of the invention, in particular the water soluble compositions, may additionally contain water soluble proteins injectable in body fluids without exhibiting any pharmacological activity at the concentrations used in the single unit dosage form of the invention. As the water soluble protein, serum albumin, globulin, collagen and / or gelatin are preferred. Such proteins may be added in amounts conventionally used in injectable pharmaceutical compositions. Thus, for example, the weight ratio of water soluble protein to BPC peptide salt is about 0.0001: 1 to 100: 1, preferably about 0.001: 1 to about 10: 1, more preferably about 0.01: 1 to about 1: 1.

The invention also relates to compositions which contain the aforementioned BPC peptide salts themselves and compositions containing them, in particular in dry and / or pure form or in aqueous or aqueous / alcoholic solutions. The pH of the solution prepared from the peptide salt or water-soluble composition of the present invention should not have any detrimental effect on the activity of the pharmacologically active peptide, but should be within an acceptable range for injection, and in addition, the pH should be used to determine the viscosity of the solution. It should be a pH that does not change significantly and does not form sediments or the like. Therefore, the pH of the solution should be about 6-9, preferably 6.5-7.5.

When converting the water-soluble composition of the present invention into an aqueous solution for administration, the concentration of the pharmacologically active peptide in such a solution is preferably about 0.0000001 to 10% (w / v), more preferably about 0.000001 to 5% (w). / v), most preferably about 0.00001 to 1% (w / v).

The composition of the present invention should preferably be in the form of a unit dosage form which further contains together the pharmacologically active BPC peptide salts of the invention and, if necessary, additives such as the aforementioned water soluble proteins. Thus, for example, by dissolving or suspending in sterile water or sterile physiological saline, the aforementioned two or three components are formed in ampoules or vials. In such cases, the method of preparation may comprise mixing a solution of physiologically active BPC peptide salt and additionally an additive solution if necessary, or adding an additive in powder form to the pharmacologically active BPC peptide salt solution, or by any other suitable technique. It can be used in combination. Dosage formulations may also be prepared by adding sterile water or sterile physiological saline to the pharmaceutically active BPC peptide salt and, if necessary, the freeze or vacuum dried powder in which the additive coexists. Such unit dosage forms may include pH adjusting agents (e.g. glycine, hydrochloric acid, sodium hydroxide), local anesthetics (e.g. xylocaine hydrochloride, chlorobutanol), isotonic agents (e.g. sodium chloride, mannitol, sorbitol), emulsifiers, adsorption inhibitors (e.g. , Conventional additives such as Tween 60 or 80), talc, starch, lactose and tragacanth, magnesium stearate, glycerol, propylene glycol, preservatives, benzyl alcohol, methylhydroxy benzoate and / or peanut hydrogen It can contain 1 or more types. Such unit dosage forms may further contain pharmaceutically acceptable excipients such as polyethylene glycol 400 or dextran.

The water-soluble composition of the present invention is preferably ingested in the formulation of a parenteral preparation. As the above-mentioned parenteral preparations, injectable solutions, transmucosal administration solutions, nasal solutions, ear solutions, and the like are preferable.

Examples of such injectable solutions include intravenous, subcutaneous, intraarterial, intramuscular and intraocular solutions. Such long lasting formulations can be easily withdrawn from the ampoules or vials with a syringe. If bubbles form when the liquid is drawn into the syringe, the bubbles can be easily removed by simply leaving the liquid for a short time.

The composition of the present invention may be a formulation dissolved in water or a lyophilized formulation with a crystallized solute such as mannitol. Sterile water or sterile saline is added to the lyophilisate to obtain an aqueous solution.

The aqueous solution osmolarity of the water-soluble composition of the present invention should be included within the acceptable range at the time of administration, and is controlled using isotonic agents such as, for example, sodium chloride and mannitol. The osmotic degree is preferably 0.5 to 2 times the osmotic degree of physiological saline, and more preferably 3/4 to 1.5 times the osmotic degree of physiological saline.

The viscosity of the aqueous solution of the water-soluble composition of the present invention should be low enough to enable injection. The viscosity is preferably less than 500 cP, more preferably less than 400 cP. Viscosity value corresponds to the value measured using Cone LD at 25 degreeC in E type viscometer (TOKIMEC, Japan).

When the composition is in the form of a lyophilizate, the viscosity, osmotic pressure and concentration of the components in the aqueous solution derived therefrom are each included in the above-mentioned ranges.

The composition of the present invention is prepared by mixing these components in a conventional manner. The purpose of mixing the components of the composition of the present invention is to maintain the activity of the pharmacologically active BPC salt and to minimize the formation of foam during the treatment. These ingredients are placed in containers (eg bottles or drums) simultaneously or in any order. The atmosphere in the container can be, for example, sterile cleaned air or sterile cleaned nitrogen gas. The resulting liquid can be transferred to small vials or ampoules, which can be further freeze dried.

Lyophilized powder formulations or liquid formulations of the compositions of the invention are biodegradable polymers such as poly (lactic acid-glycolic acid) copolymers, poly (hydroxybutyric acid), poly (hydroxybutyric acid-glycolic acid) copolymers, or these After dissolving or dispersing in a solution of the mixture of, it can be formulated, for example, into a film, microcapsules (microspheres) or nanocapsules (nanomicrospheres), in particular soft capsules or hard capsules. .

In addition, the compositions of the present invention encapsulated in liposomes comprising phospholipids, cholesterol or derivatives thereof may be further dispersed in physiological saline or a solution of hyaluronic acid dissolved in physiological saline.

Soft capsules may be filled with liquid formulations of the compositions of the present invention. Hard capsules may be filled with the lyophilized powder of the composition of the present invention, or in the case of rectal or oral administration, respectively, the lyophilized powder of the present invention may be compressed into tablets.

Of course, the composition of the present invention may be supplied in a prefilled syringe for self administration.

The composition of the present invention may be stored at a normal temperature, such as +10 ° C to +30 ° C or at a normal refrigeration temperature range, preferably about +2 ° C to +8 ° C.

The invention also relates to the novel uses of the salts and / or compositions described above, and in particular to disorders associated with nitric oxide (NO) formation or impaired NO-based function, in particular hypertension, angina, impotence, circulatory system, sepsis, Seizures, infections, respiratory disorder syndrome, adhesions and aggregation of platelets and leukocytes, endothelial dysfunction, gastrointestinal damage, peristaltic disorders, diabetes, pancreatitis, hypotension and Parkinson's disease; Dysfunction or hyperfunction of the sensory sensory nerves, especially sensory neuropathy, herpes neuralgia, atopic dermatitis, healing of damaged tissues, urticaria, psoriasis, bullous stool, eczema, photodermatitis, acquired cold or high temperature Chronic arthritis, gastrointestinal damage, specific or nonspecific reactive hyperactivity of the upper and lower airways (asthma, rhinitis); Endothelial disorders; Wounds, ulcers; Conditions associated with acute infection and / or chronic infection, in particular chronic arthritis, diseases associated with delayed type hypersensitivity, and gastrointestinal damage; Liver disease, especially organ damage caused by free radicals specifically caused by light irradiation; Diseases associated with catecholaminergic disorders, in particular schizophrenia, amphetamine administration effects, drug abuse; Stress related symptoms; Acute pancreatitis with additional positive efficacy in concomitant gastroduodenal pathology; Cardiac disorders, in particular antiarrhythmic, antiangina and cardioprotective treatments; Congestive disorders; Parkinson's disease and Parkinson's disease-like pathology; Temperature dependence disorder; Bone damage; Damage to various organs due to hypertension; Coagulation disorders; Pain disorders; Convulsive disorders; Spinal cord injury; Alcoholic damage due to alcohol abuse or increased alcohol intake; Cerebral ischemic disease; Peripheral nerve damage; Celiac disease and neuroleptic disorders; Diseases associated with abnormal or mutated lymphocytes; Fetal disorders; Osteoporosis and vaginal atrophy due to ovarian resection procedure; tumor; Viral diseases, in particular AIDS or ARC; Gastrointestinal damage; Cognitive disease; Withdrawal disorders; The present invention relates to a therapy using the salts and / or compositions described above for the treatment of diseases associated with renal disorders and cellular immune response disorders.

The present invention also relates to the aforementioned BPC peptide salts or pharmaceutical or diagnostic compositions having the following formula II, wherein the formula II is as follows.

The present invention relates in particular to a pharmaceutical or diagnostic composition comprising a BPC peptide salt or a salt of a BPC peptide according to Formula (I)

(Called BPC157)

And It is selected from the group consisting of.

Such peptides are disclosed in WO94 / 11394 and WO93 / 24521, which are incorporated herein by reference in connection with the preparation of the invention and the use of BPC peptides.

In addition, the peptide may have other functions such as carbohydrates, fats, proteins or peptides, antibodies, receptors, hormones, cytotoxic substances, marker substances, dyes, radiolabels, immunomodulators, drugs, carriers, targeting or signaling substances, and / or the like. Or to structural parts.

The invention also relates to the BPC peptide salts described above and to the compositions, wherein the peptide is linear or cyclic, in particular cyclized by an amide bond between the first and last amino acid residues.

The invention will be described in detail by the following examples and the accompanying drawings.

Example 1

Preparation of Monosodium Salt of BPC157 (NaBPC157)

0.5 g (0.35 mmol) of pentadecapeptide having the sequence Gly Glu Pro Pro Gly Lys Pro Ala Asp Asp Ala Gly Leu Val (BPC157) was dissolved in 10 ml of water containing 29.6 mg of sodium hydrogencarbonate (0.35 mmol), and 0.2 After sterile filtration with a μ filter, lyophilization was followed to obtain 0.48 g of an off-white solid.

Purity of obtained salt: 99.4% (HPLC)

Mass spectrum (FAB): 1419, 1441 (M ⁺ Na ⁺ ) (multiple ions)

Peptide salts obtained after 72 hours of hydrolysis with 6 N HCl in sealed in vitro at 110 ° C. were subjected to amino acid analysis to obtain values corresponding to compositions 3Gly, 4Pro, 2Ala, 2Asp, Glu, Leu, Val, Lys.

IR spectrum (KBr): FIG. 1

UV spectrum (H ₂ O): λ _max = 190 nm, no other peak peak

mp: 288-290 ° C (decomposition)

Example 2

Preparation of disodium salt of BPC157 (Na ₂ BPC157)

0.5 g (0.35 mmol) of BPC157 was dissolved in 10 mL of ethanol. 0.5 mol of 1.4 ml per liter of methanolic sodium hydroxide was added at moderate speed with stirring. This solution was sterile filtered through a 0.2 μ filter and slowly added to stirred diethyl ether (50 mL). The white solid separated off was filtered and washed with diethyl ether to obtain 0.52 g of disodium salt.

Purity of obtained salt: 99.4% (HPLC)

Mass spectrum (FAB): 1419, 1441 (M ⁺ Na ⁺ ) (multiple ions), 1464 (M ⁺ Na ₂ ⁺ )

Amino acid analysis of the obtained peptide salt yielded values corresponding to the composition 3Gly, 4Pro, 2Ala, 2Asp, Glu, Leu, Val, Lys.

IR spectrum (KBr): FIG. 2

UV spectrum (H ₂ O): λ _max = 190 nm, no other peak peak

mp: 275-277 ℃

Example 3

Preparation of the cesium salt of BPC157 (Cs ₂ BPC157)

0.5 g (0.35 mmol) of BPC157 was dissolved in 8 ml of water containing 114 mg (0.70 mmol) of cesium carbonate, and the solution was sterilized and filtered through a 0.2 µ filter, followed by lyophilization to obtain 0.55 g of an off-white solid.

Purity of obtained salt: 99.3% (HPLC)

Mass spectrum (FAB): 1419, 1551 (M ⁺ Cs ⁺ ) (multiple ions), 1684 (M ⁺ Cs ₂ ⁺ )

Amino acid analysis of the obtained peptide salt yielded values corresponding to the composition 3Gly, 4Pro, 2Ala, 2Asp, Glu, Leu, Val, Lys.

IR spectrum (KBr): FIG. 3

UV spectrum (H ₂ O): λ _max = 190 nm

mp: 268 ℃

Example 4

Preparation of TRIS Salt (TRIS-BPC157) of BPC157

0.5 g (0.35 mmol) of BPC157 was dissolved in 10 ml of methanol containing 42.6 mg (0.35 mmol) of tris- (hydroxymethyl) aminomethane (TRIS), and the solution was sterilized and filtered through a 0.2 µ filter. Methanol was evaporated to dryness in vacuo to afford 0.56 g of a white solid.

Purity of obtained salt: 99.5% (HPLC)

Mass spectrum (FAB): 1419 (MH ⁺ )

Amino acid analysis of the obtained peptide salt yielded values corresponding to the composition 3Gly, 4Pro, 2Ala, 2Asp, Glu, Leu, Val, Lys.

IR spectrum (KBr): FIG. 4

UV spectrum (H ₂ O): λ _max = 190 nm

mp: 250 ° C (decomposition)

Example 5

Preparation of Di-TRIS Salt [(TRIS) ₂ -BPC157] of BPC157

Compounds were prepared by the method described in Example 1 except that TRS 85.2 mg (0.70 mmol) was used.

Purity of obtained salt: 99.5% (HPLC)

Mass spectrum (FAB): 1419 (MH ⁺ )

Amino acid analysis of the obtained peptide salt yielded values corresponding to the composition 3Gly, 4Pro, 2Ala, 2Asp, Glu, Leu, Val, Lys.

IR spectrum (KBr): FIG. 5

UV spectrum (H ₂ O): λ _max = 190 nm

mp: 188-193 degreeC

Example 6

Preparation of 2-aminopropanol Salt (2-AMP-BPC157) of BPC157

Compounds were prepared by the method described in Example 1. 2-aminopropanol (2-AMP) was used as the base.

Purity of obtained salt: 96.6% (HPLC)

Mass spectrum (FAB): 1419 (MH ⁺ )

Amino acid analysis of the obtained peptide salt yielded values corresponding to the composition 3Gly, 4Pro, 2Ala, 2Asp, Glu, Leu, Val, Lys.

IR spectrum (KBr): FIG. 6

UV spectrum (H ₂ O): λ _max = 190 nm

mp: 158 ° C (decomposition)

Example 7

Preparation of Triethanolamine Salt (TEAM-BPC157) of BPC157

Compounds were prepared by the method described in Example 1. Triethanolamine (TEAM) was used as the base.

Purity of obtained salt: 99.2% (HPLC)

Mass spectrum (FAB): 1419 (MH ⁺ )

Amino acid analysis of the obtained peptide salt yielded values corresponding to the composition 3Gly, 4Pro, 2Ala, 2Asp, Glu, Leu, Val, Lys.

IR spectrum (KBr): FIG. 7

UV spectrum (H ₂ O): λ _max = 190 nm

mp: 202-205 ° C

Example 8

Preparation of Tablets Containing TRIS-BPC157

Furtherance Mg / tablet TRIS-BPC157 0.5 Trehalose 20.0 Lactose 17.0 Starch 6.5 talc 3.0 Tragacanth 2.5 Magnesium stearate 0.5 50.0 mg

TRIS-BPC157 (0.5 mg) and trehalose (20 mg) were dissolved in 1 ml of water and dried by evaporation. After completion of drying, the crude residue was mixed with the other ingredients to make a tablet.

Example 9

Preparation of Capsules Containing NaBPC157

Furtherance Mg / capsules NaBPC157 0.5 Trehalose 60.0 Lactose 39.0 Magnesium stearate 0.5 100.0 mg

NaBPC157 (0.5 mg) and trehalose (60 mg) were dissolved in 1 mL of water and the solvent was removed by evaporation. The crude residue was mixed with the other ingredients to make a capsule.

Example 10

Preparation of a Liquid Containing NaBPC157

Furtherance g / 25 ml NaBPC157 0.05 Glycerol 15.00 Benzyl alcohol 0.01 Added pH 7.0 buffer 25 ml

Example 11

Preparation of a Cream Containing TRIS-BPC157

Furtherance g / 25 g TRIS-BPC157 0.05 Emulsifier 2.80 Hydrogen peanut oil 7.08 Trademark Tween 60 12.08 Propylene glycol 3.00 Methylhydroxy benzoate 0.07 25.00 g

Example 12

Stability test

The salts were incubated at 40 ° C. for 76 and 120 days to test the stability of the BPC peptide salts. The concentration of BPC peptide salt in the aqueous solution was 0.2% (w / v). HPLC method, ie Kromasil 100 column, 5 μ, 150 × 4.6 mm, 0.1% (0-50% by volume) of trifluoroacetic acid in mobile phase water / acetonitrile, gradient elution for 25 minutes, flow rate 1 ml / min, detection UV 214 nm.

For comparative example, free BPC peptide and its monoacetate were used.

Assay of stability area ratio (%) of BPC peptide salt at 40 ° C. on day 0, 76 and 120 (HPLC) compound pH value (0.2% in water) Day 0 Day 76 Day 120 Free BPC157 peptide 4.46 99.3 98.1 97.4 BPC157 Peptide Monoacetate 4.53 99.2 97.8 95.3 Sodium salt of BPC157 6.51 99.4 99.5 99.4 Disodium salt of BPC157 7.64 99.4 99.7 99.4 TRIS salt of BPC157 6.31 99.5 99.5 99.4 Di-TRIS Salts of BPC157 8.24 99.5 99.5 99.5 2-AMP salt of BPC157 8.20 99.6 99.4 99.5 TEAM salt of BPC157 7.61 99.2 99.3 99.1

The data in Table 1 shows that the stability of the BPC peptide salt of the present invention was increased than that of free BPC peptide or monoacetate thereof. It is also believed that the injection of these solutions does not cause pain or necrosis due to the high pH values of the BPC peptide salt solutions.

As another experiment, it can be seen that the stability of the BPC peptide salts of the crude and liquid formulations of the present invention was further improved after the addition of trehalose. Therefore, addition of trehalose as a pharmaceutically acceptable additive for the preparation of pharmaceutical compositions, especially tablet or capsule formulations, is another important feature of the present invention.

The following examples illustrate the experiments illustrating the pharmacological activity of the BPC peptide salts of the invention. In these experiments a number of conventional models were performed in an "in vitro" or "in vivo" test. For these experiments, the monosodium salt of BPC peptide 157 (abbreviated as NaBPC157) was used unless otherwise noted. Unless otherwise noted, male Wistar rats weighing 250-280 g were used for all experiments.

Example 13

NO-based

Introduction

Nitric oxide (NO) acts as a signaling molecule in endothelial and neuronal cells and as a killer molecule activated by immune cells. Recent studies have reported that it can be used as a medicament by inhalation. In general, NO is caused by excessive damage to various disorders, especially hypertension, angina, erectile dysfunction, circulatory system, sepsis, seizures, infections, respiratory disorder syndrome, pulmonary hypertension, adhesions and aggregation of platelets and leukocytes, diabetes, hypotension and It has been reported that it can act on Parkin's disease.

Materials and Techniques Used

Several effects, such as the activity of NaBPC157 and beneficial activity on gastrointestinal damage with respect to blood pressure maintenance, were tested by administering NaBPC157 (10 μg or 10 ng / kg) to rats. Damage was obtained using ethanol (96%, stomach) for 1 hour. NaBPC157 was administered at the same time (intraperitoneal). In blood pressure maintenance experiments, NaBPC157 was administered intravenously.

In addition, NO-precursor L-arginine (200 mg / kg intravenously) (D-arginine is ineffective) and N ^G -nitro-L-arginine methylester, a competitive inhibitor of endothelial nitric oxide (NO) production NAME) (5 mg / kg intravenously) was tested. In gastrointestinal damage analysis, NO- formulations were administered 5 minutes prior to treatment with ethanol damage and NaBPC157.

result

In the ethanol test, administration of NaBPC157 alone has the same anti-ulcer efficacy as L-arginine. NaBPC157 inhibits the severe gastrointestinal damage observed in ethanol treated control rats. L-NAME was ineffective. L-NAME completely inhibited the activity of L-arginine, but only weakened the activity of NaBPC157. After administration of the combination of L-NAME + L-arginine, the activity of NaBPC157 was further reduced.

In the blood pressure study, NaBPC157 (no effect on baseline standard) compared to L-arginine was found to mimic the effect [the L-NAME blood pressure increase was reduced, administered prophylactically, and the maximum L-NAME blood pressure increase (ie, after L-NAME). Already increased L-NAME value at 10 minutes) and preventive activity (L-arginine induced moderate blood pressure reduction is inhibited by NaBPC157 pretreatment). If NaBPC157 is administered 10 minutes after the combination of L-NAME + L-arginine, which still increases blood pressure, its pronounced efficacy (appears in L-NAME treated rats) disappears. In gastric mucosal in vitro experiments from rat gastrointestinal tissue equivalents, NaBPC157 at the same dose (100 μM) as L-arginine forms large amounts of NO. However, the efficacy of NaBPC157 cannot be inhibited by L-NAME, even if it uses an amount 10 times (100 vs 1000 μM) than the required amount of L-arginine efficacy inhibition. In contrast, NO synthesis is reduced when using NaBPC157 and L-arginine in combination. In summary, NaBPC157 can inhibit NO-efficiency in both specific aspects of gastrointestinal mucosal integrity and blood pressure maintenance, particularly when used in combination with L-arginine, which is highly efficacious and / or very unusual for NO. It can be seen that.

Since NaBPC157 has a more potent effect on NO than L-arginine, it can prevent excessive NO formation associated with side effects [inhibited potency of L-arginine (ie, hypotension)]. These side effects reverse normal values in vivo, and excessive NO formation is inhibited in vitro. It also inhibited the negative consequences of NO-based inhibition (ie, prevention of L-NAME induced blood pressure increase and reversal of existing L-NAME hypertension).

Based on the close similarity of NO analysis in other tissues (e.g. lung, liver, blood vessels, etc.) and the diversity of the models used (gastrointestinal damage and blood pressure maintenance), It has been reported that it has beneficial efficacy. In particular, the BPC peptide salts of the present invention are hypertension, angina, erectile dysfunction, circulatory system, sepsis, seizures, infection, respiratory disorder syndrome, pulmonary hypertension, pancreatitis, adhesion and aggregation of platelets and leukocytes, endothelial dysfunction and Parkins It is useful for the treatment of diseases.

Example 14

Sensory neurons

Introduction

Somatosensory neurons are generally associated with the regulation of bioalwaysty, particularly in response to aggression. Such neurons can detect potential treatment. Neurons can immediately initiate appropriate measures to mitigate risk by themselves. Thus, vascular afferent neurons represent the first week of defense against trauma. In general, this protection has been demonstrated by experimental damage to the skin and gastrointestinal mucosa. Dysfunction or hyperactivity is caused by various diseases, especially congenital sensory neuropathy, sensory neuropathy due to diabetes mellitus, shingles, postherpetic neuralgia, atopic dermatitis, and healing of damaged tissue (e.g., persistent skin wounds, acids). Exacerbation of skin damage, the formation of keratitis-like damage in the cornea), urticaria for acquired cold and high temperature, psoriasis, bullous dermatitis, eczema, photodermatosis, diseases of the upper or lower airways, specific or nonspecific reactive hyperactivity, Vasomotor neuronitis, asthma, chronic arthritis and gastrointestinal damage.

Materials and Techniques Used

The gastroprotective efficacy of NaBPC157 on gastrointestinal damage (formed by treatment with rats with 96% ethanol, bondage stress and indomethacin treatment) was tested. The possible relationship of sensory neurons in the beneficial efficacy of NaBPC157 (10 μg / kg, 10 ng / kg intraperitoneal) was tested using capsaicin, which has unique effects on sensory neurons, and is an animal adult (3 months old). Administration at high doses (125 mg / kg, subcutaneously) or newborn animals (7 days old) (50 mg / kg, subcutaneously) destroys sensory fibers, while low doses (500 μg / kg, peritoneal) Internally) activating the release of neurotransmitters and the protective efficacy of the mucosa.

result

(i) Without capsaicin, NaBPC157 protects the gastrointestinal mucosa from ethanol, bondage stress and indomethacin.

(ii) When the neurotoxic dose of capsaicin is administered, the deleterious effects of capsaicin on bond stress, ethanol or indomethacin damage have a significant effect on the beneficial activity of NaBPC157. The protection of NaBPC157 was evident in all of these assays in models treated with capsaicin (both adult and neonatal). After neoplasia treatment with capsaicin, gastrointestinal protective effects caused by NaBPC157 have been reported to disappear completely when NaBPC157 is administered as a single ng regimen. When the same dose was used daily the mucosal protective effect was completely reversed.

(iii) With respect to the excitatory administration of capsaicin, the beneficial activity of NaBPC157 is further increased.

These data illustrate the complex synergy between the beneficial activity of NaBPC157 and the peptidic sensory afferent neuronal activity. Given the efficacy of capsaicin in animals and humans and the close similarity of the foregoing experiments, NaBPC157 can be used to treat the aforementioned disorders.

Example 15

Endothelial protection

Introduction

Vascular endothelial injury occurs prior to the progression of visible organ damage and occurs essentially with it. Endothelial protection can reduce ischemic damage to mucosal integrity. As a useful model, the administration of Monastral Blue (MB; Sagmar Company, USA) just prior to administration of necrotic agents (e.g. ethanol administered to the stomach), which is known to cause significant damage to rats, is widely accepted and used. .

Materials and Techniques Used

All rats were administered IV intravenously 3 minutes before ethanol treatment. Rats were killed 1 minute after ethanol treatment. NaBPC157 (10.0 μg / kg, intraperitoneal) or saline (5.0 mL / kg intraperitoneally) was administered 1 hour prior to ethanol treatment. Immediately after the rats were killed, the stomach was removed and the observers without preconceived notions assessed the extent of the injury. Representative sites of the stomach and duodenum were processed for further histological analysis.

Vascular injuries were assessed early (one minute after ethanol treatment) using the technique using MB. The site density of the colored mucosa was examined by TEM.

result

MB staining was significantly reduced in the group treated with NaBPC157. The close similarity to the model used above with respect to the human case illustrates the use of NaBPC157 in the treatment of conditions associated with endothelial disorders in human treatment.

Example 16

Angiogenesis

Introduction

Angiogenesis is very important for the formation of granulation tissue and for healing wounds and / or ulcers. Angiogenic properties were tested using commonly known methods.

Materials and Techniques Used

In each rat, an equivalent amount of NaBPC157 (50 μg, 10 μg, 10 ng / ml) or related agent, cimetidine (10 mg, 100 mg, 500 mg / ml), ranitidine (2.5 mg, 25 mg, 250 mg / ml) Ml), two sterile sponges (1 cm × 1 cm × 0.25 cm) containing pamotidine (10 mg, 50 mg, 100 mg / ml) and sucralate (1 mg, 5 mg, 10 mg / ml) (V = 0.25 mL)] was implanted subcutaneously. Sponges were removed after 3 and 7 days. It was fixed in formalin and processed for histological evaluation, histochemical evaluation and biomorphometry. The microscope used was a program "SFORM" manufactured by VAMS, Leitz, DIAPLAN, Zagreb, Croatia, for biomorphometric analysis.

result

Newly formed granulation tissue around the implanted sponge is used regularly as a meaningful quantitative measure of the graft response to foreign bodies. Experiments using rats that were killed three days after transplantation yielded the following results. More granular tissue was observed in the animals treated with NaBPC157 compared to the control. Similar results were observed in rats treated with all doses of sucralate used. In contrast, no differences were observed between control and analytical formulations in the groups treated with all three H2 blockers. In animals killed at 7 days after transplantation, animals treated with all three doses of sucralate and animals treated with the highest amount of NaBPC157 (50 μg) showed a significant difference compared to the control group. The control group did not differ significantly between the two periods of killing the animals.

The endothelial space was counted inside the newly created granulation tissue. Regarding the value of the control group (newly generated endothelial space in the control group at day 3 post transplantation was 7.94 ± 1.23 and 14.8 ± 3.12 at day 7 after transplantation), all the materials used were in these two periods (day 3 and Significantly increased in all cases).

In addition to the fact that various anti-ulcer drugs share angiogenic properties, NaBPC157 stimulates granulation like sucralate. Thus, NaBPC157 initiated and demonstrated efficacy in the healing process, in particular the wound and / or ulcer healing process.

Example 17

infection

Introduction

Anti-inflammatory agents that have been used until recently are typically evaluated in many suitable models that detail human acute and / or chronic infection disorders. However, severe gastrointestinal damage appeared as a major side effect of these formulations.

NaBPC157 [e.g., terpentine, carrageenan, acetic acid or MgSO ₄ , writhing (prostaglandin dependent, prostaglandin independent) tail tingling with antipyretic efficacy [reduction of fever induced by yeast (4000 mg / kg subcutaneous)] tail-pinching test] has been reported to have acute anti-inflammatory and analgesic activity. Therefore, its efficacy as a nonsteroidal anti-inflammatory agent in NSAIA-induced gastrointestinal injuries and efficacy against chronic inflammatory injury as adjuvant arthritis, beneficial known efficacy for gastrointestinal damage, was simultaneously tested using rats.

Materials and Techniques Used

In the gastrointestinal injury [indometacin (30 mg / kg subcutaneous), aspirin (400 mg / kg gastric) and diclofenac (125 mg / kg intraperitoneal)] experiment, NaBPC157 (10 μg or 10 ng / kg intraperitoneal) was simultaneously Or 1 hour prior to drug (indomethacin) administration. Adjuvant arthritis (0.2 ml of Freund's Supplement to the tail) In experiments (14 days, 30 days, 1 year), a single dose of NaBPC157 (10 μg or 10 mg / kg intraperitoneally) (before and after Freund's Supplement) 1 Time) or once daily (0-14 days, 14-30 days, 14 days-1 year).

result

Administration of NaBPC157 with the tested NSAIA significantly reduced damage to the small intestine in the indomethacin group, as well as marked damage in the gastrointestinal tract of control rats. In the adjuvant arthritis experiments, the progression of the injury was greatly reduced after a single dose of NaBPC157, and was more alleviated in rats treated with NaBPC157 daily. In conventional adjuvant arthritis treatment, the beneficial efficacy of NaBPC157 appears only two weeks after drug administration, which becomes more apparent one year after administration. These data demonstrate the anti-inflammatory and protective efficacy of NaBPC157 on mucosal integrity.

Two distinct mechanisms of inflammation and delayed hypersensitivity are known to be involved in the regulation of adjuvant arthritis. NaBPC157 has beneficial effects on both of them. In the context of reference drugs (eg, aspirin, indomethacin), NaBPC157 shows significant efficacy even at small doses (μg and ng / kg vs mg / kg). The initial efficacy of this adjuvant arthritis prevention is even greater with daily administration. Large doses (10 μg / kg) show efficacy after a single or daily regimen, whereas small doses (10 ng / kg) do not show efficacy with a single dose, and can be administered with a daily regimen. Only when it shows efficacy. These findings proved to be useful dosing during the full progression of adjuvant arthritis in relation to the therapeutic effect of NaBPC157 in fully advanced arthritis. It can be seen that the reported efficacy of NaBPC157 in adjuvant arthritis (prophylactic / therapeutic efficacy, existing / existing adjuvant arthritis) cannot be obtained using currently used therapeutics. Daily administration of glucocorticoids is effective for the prevention of adjuvant arthritis, but not for short-term treatment. Immunosuppressants (high doses) and nonsteroidal analgesics show efficacy only in pretreatment or aftertreatment respectively.

Therefore, it has been demonstrated that NaBPC157 with mucosal protective properties can be used for the treatment of conditions associated with acute inflammation and chronic arthritis based on the close similarity between the model used and the disorder of the person concerned. Thus, NaBPC157 can be used in the treatment of diseases associated with delayed types of hypersensitivity disorders and gastrointestinal damage caused in various sites of the gastrointestinal tract, in particular those caused by agents such as NSAIA.

Example 18

Free radical scavengers efficacy

Materials and Techniques Used

The hepatoprotective efficacy of NaBPC157 was compared with reference drugs such as bromocriptine, amantadine and somatostatin with 24-hour bile duct and hepatic artery ligation, 48-hour restraint stress, and CCl ₄ (free radical stimulator) administration in various experimental models of liver damage in rats. Evaluated. NaBPC157 was administered intragastric or intraperitoneally.

result

NaBPC157 significantly inhibited the progression of liver necrosis or fat change in rats treated with 24 hours bile duct and hepatic artery ligation, 48 hours restraint stress and CCl ₄ treatment (1 ml / kg intraperitoneal, then 48 hours later). Other reference drugs showed little or no protective efficacy in this model. Laboratory tests on bilirubin show that SGOT and SGPT are fully correlated with visual / microscopic findings. Thus, NaBPC157 can be used to treat liver liver disease. In addition, myelosuppression by light irradiation becomes a reproducible model for experimenting the repair kinetics of hematopoietic components. NaBPC157 has a beneficial effect on bone marrow damage caused by sub-lethal light irradiation.

Therefore, NaBPC157 may be effective for the treatment of bone marrow damage caused by light irradiation. Given the progression of damage and the production of free radicals and the generally known relationship of CCl ₄ , the effective efficacy of NaBPC157 has also been shown to be administrable to other free radicals, particularly organ damage caused by light irradiation.

Example 19

Catecholaminetic system

Introduction

Indirectly acting sympathetic drugs, such as amphetamines, have common properties, such as causing both inhibition of catecholamine reuptake (primarily dopamine) and increased catecholamine release at the nerve endings in the central nervous system (CNS). Homologous behavior is manifested by activation of the dopaminergic system in the striatum. In general, the increase in the amphetamine climbing pattern is due to the upregulation of the streptococcal dopamine receptor following the administration of doperamine antagonist haloperidol. This leads to amphetamine supersensitive progression. Administration of NaBPC157 does not affect the behavior identified by the naked eye or cause homology.

Materials and Techniques Used

The efficacy of NaBPC157 on dopamine agonist amphetamine (10 mg / kg intraperitoneal) homology and excitability was examined. NaBPC157 was administered as a beneficial regimen (10 μg or 10 ng / kg intraperitoneal) for prophylactic concurrent treatment or treatment. Homologous behavior and excitability were induced in mice.

result

Significant alleviation and reversal (dose at the maximum of amphetamine disorders) in both homologous behavior and increased excitability (ie, stronger and more intense cramps, sudden jumps and escape) appeared regularly.

Note that NaBPC157 affects increased climbing behavior in mice. Mice were pretreated with dopamine antagonist haloperidol (5.0 mg / kg intraperitoneal) and then treated with amphetamine (20 mg / kg intraperitoneally, 1, 2, 4 and 10 days after haloperidol pretreatment), which is usually Amphetamine stimulation efficacy was used in behavioral hypersensitivity experiments. Thus, the antagonism of amphetamine homology appears as a result of the dopamine antagonistic activity (direct or indirect) of NaBPC157 tested, with the potential for increased efficacy of haloperidol on amphetamine climbing behavior. In contrast, co-administration of NaBPC157 with haloperidol showed almost complete reaction. This date also demonstrated the interaction of NaBPC157 with the dopamine system. In addition, the interaction between central dopamine and NaBPC157 was also demonstrated in other experimental models (ie, prevention of stress ulcers). Interactions with the dopamine system have already been reported for many known peptides (neurotensin, CCK, etc.). In addition, administration of NaBPC157 likewise reverses behavioral disorders caused by LSD (eg, 0.3 mg / kg intraperitoneal). NaBPC157 has regulatory efficacy in the dopamine system. In the state of increased dopamine release and synthesis induced by amphetamines, NaBPC157 can inhibit and reverse the resulting disorders (eg, homologous behavior). Similarly, NaBPC157 can greatly attenuate the consequences of dopamine receptors blocked by haloperidol. This also suggests that the regulatory efficacy of NaBPC157 replaces the dopamine system, which is significantly insufficient. This eliminates hypersensitivity of dopamine receptors and increased amphetamine disorders.

Therefore, NaBPC157 is a useful agent for the treatment of schizophrenia, amphetamine administration effect (schizophrenia from psychosis) and drug abuse.

Example 20

stress

Introduction

Stress is defined as a nonspecific state caused by damage in various organs. This stress response is defined as a response to various harmful conditions. Most commonly used in animal stress models are bond stress models that cause severe stress gastrointestinal damage in rats. Other organs may also be affected.

Substances used, techniques and results

NaBPC157 strongly inhibited the inevitable progression of severe gastrointestinal damage by administering 10 μg or 10 ng / kg body weight, intraperitoneal or intragastric doses 1 hour prior to stress induction. If the time between stress relief administration and stress induction (hereinafter referred to as "duration") is prolonged, the standard antiulcer drug is substantially ineffective, which contrasts with the apparent efficacy of the tested NaBPC157. NaBPC157 retained beneficial activity even after an extended period of 48 hours. The protection conferred by NaBPC157 includes the alleviation of the damage regularly present in other organs (eg, liver, adrenal gland, kidney, testes, heart, pancreas, spleen). Thus, it is clear that other commonly known stress variables are less disturbed (ie, thymic lymphatic degeneration and cortical hypertrophy of the adrenal gland), thus NaBPC157 can be administered to a variety of stress states. NaBPC157 has beneficial benefits for various injuries that regularly appear in various organs in connection with nonspecific stress pathology. Given the same associations as in humans, as in the model used, this property is beneficial because NaBPC157 is beneficial even when stress pathology is already in progress (eg, 24 hours after stress induction). It is emphasized more.

Example 21

Demonstrate cytoprotective efficacy

Introduction

Cytoprotection is inherently defined as a property that protects cells from various harmful agents, and has the effect pointed out in the gastric mucosa as an independent potency of gastric acid. This definition was later interpreted broadly to include nearly similar protective (cytoprotective-organic) efficacy outside the gastrointestinal tract, including damage to various organs. The cytoprotective efficacy achievable by administering NaBPC157 was tested based on their effects in the early Roberts model on cytoprotectants that select for ethanol-induced gastrointestinal damage.

Substances used, techniques and results

NaBPC157 (10 μg or 10 ng / kg, intraperitoneal) has a protective effect of 96% of ethanol (1 ml per rat, intragastric) 1 hour before or concurrently with administration, and beneficial efficacy (ethanol administration 1 Administration at maximum injury progress after time).

To create an acid free environment for cytoprotective experiments, a total gastrectomy was performed 24 hours before the ulceration procedure. In the absence of gastrointestinal and gastric acid, the damaging efficacy of cysteamine (400 mg / kg, subcutaneous, killing 24 hours later) and NaBPC157 (10 μg or 10 ng / kg, so far considered to be an acid-related duodenal ulcer trigger) The cytoprotective efficacy of the intraperitoneal) is shown in reference agents known to have cytoprotective properties (cimetidine (50), ranitidine (10), operprazole (10), bromocriptine (10) and atropine (19) Mg / kg unit, intraperitoneal, 1 hour before cysteamine). In rats with untreated stomachs, the entire administered substance has a potent beneficial effect. In gastric resected animals, administration of the studied agent (ie, NaBPC157 or reference agent prior to cysteamine) strongly inhibits the severe progression of duodenal injury reported in control rats with gastric resection. In the treatment group not treated with cysteamine, there was no damage (only 24 or 48 hours after abdominal and gastrectomy surgery), and the potential injury was also seen in the treatment group treated with cysteamine in animals with abdominal wall ablation. Not observed. These findings (the same damaging effect of cysteamine, the same protective, damaging or protective effect of the reference agent and NaBPC157 in gastric resection of rats without stomach and rats with stomach) were not associated with gastric acid secretion). Indicates. A pseudotype (damage not related to gastric acid) between gastric injury (eg ethanol) and cysteamine duodenal injury is clearly shown. Acid-independent high "cell protection" is a common phenomenon for all test preparations, but has proven to be more potent for NaBPC157.

Since NaBPC157 is efficacious at much lower dosages (eg, μg or ng / kg vs mg / kg) than other formulations, this efficacy (and therapeutic effect) extends to the damage of many other organs as well. Can be. This is because the efficacy of other agents in other organs is expected to be due to their "cytoprotective" efficacy (eg, somatostatin: damage to the adrenal gland, pancreas, liver, lung and gastrointestinal tract).

Example 22

Prove institutional protection

Introduction

Endothelial protection studies are generally accepted to usefully extend "cytoprotective efficacy" to "organ protective" efficacy. For clarity, experimentation with MB in the gastric mucosa of ethanol-damaged rats is widely accepted due to the ability of MB to bind to the damaged endothelium.

Materials and Techniques Used

All rats were given MB (10 mL / kg body weight, intravenously) 3 minutes before ethanol treatment and animals were killed 1 minute after ethanol treatment. NaBPC157 (10.0 μg / kg, intraperitoneal) or saline (5.0 mL / kg, intraperitoneal) was administered 1 hour prior to ethanol treatment. Immediately after killing the animal, the stomach was removed and the observer without the preconceived view as described above evaluated the extent of the injury. Representative sites of the stomach and duodenum were processed for further histological analysis. Vascular injuries were assessed early (one minute after ethanol treatment) using the technique using MB. The site density of the colored mucosa was examined by TEM.

result

MB staining was significantly reduced in the group treated with NaBPC157. In addition, administration of NaBPC157 did not show adverse effects. Despite administration at high doses (eg g / kg body weight, intraperitoneal), there were no effects on various basic variables and no toxicity was observed.

Given the important role of endothelial protection for organ protective efficacy and the presence of endothelial vascular tissue evenly distributed throughout the body, NaBPC157 can be used as an organ-protective agent in the damage of various organs.

Example 23

Acute pancreatitis

Materials and Techniques Used

NaBPC157 was tested using rats as a therapeutic or as a protective agent for acute pancreatitis (induced by bile duct ligation). The effect of NaBPC157 on concurrent gastrointestinal and duodenal injury was investigated.

NaBPC157 (10 μg or 10 ng / kg body weight, intraperitoneal, gastric) was administered prophylactically 1 hour before ligation, whereas treatment was administered daily on day 1 after ligation (final administration 24 hours before killing). . Efficacy was examined at 1 day intervals until the last day 5 days after ligation.

result

After pretreatment administration, the pancreas was strongly protected. Clear benefit has also been achieved when administered to existing severe acute pancreatitis conditions. In assessing the appearance of necrosis, edema, neutrophils and monocytes, the more necrosis, edema and neutrophils were observed in rats treated with NaBPC157. Analyzing serum amylase levels in relation to control data reported significantly lower elevations (NaBPC157 pretreatment administration) as well as a loss of already increased levels (NaBPC157 treatment administration). In addition to the beneficial effects on pancreatitis, the beneficial effects of NaBPC157 on gastrointestinal and duodenal injury progression in rats with bile duct ligation have been shown in both pre and post treatment. NaBPC157 can be used for the treatment of acute pancreatitis with additional beneficial effects on concurrent gastroduodenal pathology.

Example 24

Efficacy on Cardiotoxicity

Materials and Techniques Used

This experiment was performed on Albino Wistar rats and Hartley guinea pigs. Barium chloride solution (10 mg / kg body weight), desipramine (10 mg / kg), digitalis (total dose 6.5 mg / kg) and isoprenin (15 mg / kg) were administered via jugular cannula Toxicity was caused. In addition, doxorubicin was administered in multiple doses (3 mg / kg, subcutaneously, once per week over 13 weeks) and in a single dose (7 mg / kg, intravenously), causing cardiomyopathy. In addition, cardiotoxicity was induced by immobilized stress as a nonpharmacologic myocardial injury. NaBPC157 was administered as shown below.

Barium Chloride

a) Pretreatment (1 hour prior): NaBPC157 50 μg / kg, 10 μg / kg and 10 ng / kg intraperitoneally

b) Post-treatment (after 60 seconds): 10 μg / kg and 10 ng / kg NaBPC157, intravenously

2. Desipramine: 50 μg / kg NaBPC157 and 50 ng / kg intraperitoneally 1 hour before

3. Digitalis: NaBPC157 50 μg / kg and 50 ng / kg intravenously at 15 minute intervals

4. Isoprenaline: 50 μg / kg NaBPC157 and 50 ng / kg intravenously 15 minutes ago

5. Immobilization Stress: 10 μg / kg and 10 ng / kg NaBPC157, intraperitoneally, 1 hour before and immediately after tying and at 24 and 48 hours of immobilization

6. (a) Chronic doxorubicin toxicity: 10 μg / kg and 10 ng / kg NaBPC157, administered simultaneously with doxorubicin, intraperitoneally

(b) Acute doxorubicin toxicity: 10 μg / kg and 10 ng / kg NaBPC157, 1 hour prior to doxorubicin administration, intraperitoneally

In the arrhythmia model, the ECG of the anesthetized animal was continuously recorded. Records were expanded to 50 mm / s or 100 mm / s every 15 seconds or when heart failure occurred. In other models, simply anesthetized animals were recorded at a recording paper speed of 50 mm / s on a repetitive or one-time enlarged record. Doxorubicin toxicity was assessed by visual and microscopic examination of other organs and hearts by biochemical analysis. A similar procedure was performed for the passivation stress test.

result

The antiarrhythmic efficacy of NaBPC157 in the arrhythmia model induced by barium chloride was observed. In pretreatment experiments, NaBPC157 delayed and inhibited the appearance of arrhythmia and reduced and / or prevented ischemia. In post-treatment experiments, NaBPC157 caused a rapid transition to venous sinus rhythms and inhibited arrhythmia reproduction.

NaBPC157 suppressed the sudden decrease in heart rate and the conduction disturbances (PQ prolongation and QRS prolongation) induced by decipramine. In addition, NaBPC157 inhibits ventricular tachycardia associated with the total venous efficacy of desipramine and severe atrioventricular obstruction. This efficacy is dose dependent.

In addition, NaBPC157 has selective efficacy against cardiac toxicity induced by digitalis. NaBPC157 exhibits beneficial efficacy for its sudden reduction as a means of inhibiting or alleviating pulse and heart rate disorders (ventricular extracontraction, ventricular tachycardia and atrioventricular obstruction). Efficacy when using NaBPC157 in ng against slowing conduction induced by toxic doses of digitalis is not significant. The efficacy was great when NaBPC157 was administered in μg.

NaBPC157 prevents ischemia and myocardial infarction. Efficacy was greater when administered at a dose of μg. ECG demonstrated that single administration of NaBPC157 in the form of pretreatment prevents ischemia and histological myocardial damage during the immobilization stress test. Ischemia was relieved by post-treatment administration of NaBPC157, which was already manifested by ECG. The use of NaBPC157 at a μg dose resulted in an increase in voltage in the QRS group, especially in pretreatment dosing.

Administration of NaBPC157 significantly reduced the pathologic findings of myocardial disorders of anthracycline (severe myocytes and vascular wall damage and fear formation) after single and multiple doses of doxorubicin. LDH activity was significantly reduced, despite significantly increased absolute levels.

In summary, NaBPC157 has proven useful as an antiarrhythmic, antiangina and cardioprotectant.

Example 25

Antidepressant activity

Materials and Techniques Used

Various antidepressants have antiulcer activity, and models commonly used in ulcer and depression studies share significant similarities. Therefore, depressive disorders are effectively controlled by primary anti-ulcer agents, which are two rat depression assays, forced swimming test (Porsolt technique) and chronic unpredictable stress technique (after 5 days of unpredictable stress protocol, Drug administration once a day during the stress technique, open field-immobilization test analysis on days 4 and 6).

result

In the forced swim test, the reduction in immobilization time of rats treated with NaBPC157 (10 μg, 10 ng, 10 pg / kg, intraperitoneal) resulted in 15 mg or 40 mg of conventional antidepressant, imipramine or nialamide, respectively ( Corresponds to the activity of the animals treated with intraperitoneal). Further deterioration of experimental conditions in chronic unpredictable stress procedures resulted in the loss of imipramine activity (30 mg), whereas the dose dependence of NaBPC157 (10 μg, 10 ng) was associated with motility in chronically stressed rats. Improved.

Example 26

Parkinson's disease model

Materials and Techniques Used

Parkinson's, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (known to destroy the dopamine black line system by forming free radicals) (30.0 mg / kg body weight, abdominal cavity 50.0 mg / kg body weight, intraperitoneal, or reserpin (catecholamine vesicle excretion) (5.0 mg / kg body weight, intraperitoneally) was administered once daily, once daily for 4 days, and after 4 days. NaBPC157 (1.50 μg or 15.0 ng / kg body weight, intraperitoneally) was administered 15 minutes before or 15 minutes after each MPTP administration. In reserpin experiments, NaBPC157 (10.0 μg or 10 ng / kg body weight, intraperitoneal) was administered 15 minutes before or 24 hours after complete stiffness after reserpin administration.

result

NaBPC157 strongly improved MPTP impaired sensory orientation and reduced MPTP induced hyperactivity. In addition, NaBPC157 reduced MPTP motor abnormalities (which are pronounced in tremors, ataxia, stiffness, especially in saline controls), nearly eliminating the normal lethal progression of MPTP treatment in the controls. In reserpin experiments, NaBPC157 strongly prevented the progression of very pronounced celiac disease. When administered 24 hours later, NaBPC157 reversed the stiffness that formed. This was observed in the reduction of reserpin hypothermia (NaBPC157 pretreatment) and the reversal of further pronounced temperature reduction (NaBPC157 posttreatment).

All of the common animal models used above are known to represent a precursor to human disorders and their treatment. Efficacy has been demonstrated in pre- and post-treatment experiments under the regimen of μg and ng and NaBPC157 has been shown to be suitable for use in the treatment of Parkinson's disease and Parkinson's disease-like pathology.

In addition, the efficacy observed in reserpin hypothermia led to the conclusion that NaBPC157 is suitable for the treatment of body temperature disorders.

Example 27

Efficacy in healing bone dysfunction

Materials and Techniques Used

The bone formation efficacy of bone marrow and TRIS-BPC157 on the healing of segmental bone insufficiency was tested in 42 rabbits during 6 weeks postoperatively. Following induction of damage (0.8 cm periosteal failure occurred in the middle of both radii), experiments with bone marrow and TRIS-BPC157 (6 rabbits per group) were performed as follows. Injured animals treated with saline (2 ml intramuscular and 2 ml topical for each bone failure) were used as controls (group 1). In groups 2 and 3, each bone failure was treated locally with 2 ml of autologous bone marrow (day 7 post surgery) or TRIS-BPC157 (10 μg / kg body weight, days 7 and 14 post surgery). In groups 4, 5 and 6, TRIS-BPC157 was administered once daily (10 μg or 10 ng / kg body weight) during the period 7 days, 9 days, 14 days and 16 days or 7 days to 21 days after surgery. It was. As standard treatment immediately after bone insufficiency formation, bone insufficiency of group 7 was filled with autocortical grafts.

All animals were killed 6 days after surgery. Biweekly x-ray and histological examinations evaluated the healing of segmental insufficiency.

result

X-ray images taken at four intervals (facial surface, microscope densitometry of bone insufficiency) and quantitative histomorphologic comparisons showed that bone marrow and intramuscularly administered TRIS-BPC157 (particularly 14 days) in bone failure The daily dose of 10 μg / kg body weight during the period significantly improved bone healing (p <0.001). This efficacy corresponds to the efficacy of autocortical grafts.

Given the clinical correlation of these experiments, bone marrow and TRIS-BPC157 (particularly the daily dose of 10 μg / kg body weight over a 14 day period) were found to have cured bone failure. Because administration is simple and the risk of complications is low, intramuscular administration of osteogenic substances such as TRIS-BPC157 may be used for other more complex surgical techniques (eg, bone grafts, angiogenic bone grafts) for treatments that heal human injury. , Ilizarov technique) is more beneficial.

Example 28

Wound healing effect

Materials and Techniques Used

NaBPC157 was used to obtain efficacy for several factors related to the healing process. Factors considered to be important in the healing process include generation of granulation tissue, angiogenesis and collagen production. The efficacy of NaBPC157 on granular tissue, collagen production and angiogenesis as well as tensile strength progression was tested using three experiments: 1) skin incision wound, 2) colon-colon anastomosis and 3) esophageal duodenal anastomosis. . Samples were histologically analyzed for collagen, reticulin, and blood vessels using cut-off and biomorphometry techniques.

result

In all experiments, the difference between NaBPC157 treated rats and controls was shown to be large. Experiments have been shown to strongly promote the invasion of NaBPC157 in the healing process. Such efficacy can be obtained by several routes of administration, including intragastric and topical administration. In addition, the analysis of tensile strength revealed an increase in levels in NaBPC157 treated rats.

Given the close similarity between the animal model and the disorder of the person concerned and the high similarity between the rat and human healing processes, NaBPC157 can be used to treat wound healing in humans.

Example 29

Efficacy on Respiratory Disorders

Materials and Techniques Used

Guinea pigs who underwent general blood flow measurement, urethane anesthesia (1.5 g / kg, intraperitoneal), artificial ventilation under constant pressure (0.98 kPa), and relaxation treatment of skeletal muscle tissue by susamethonium (0.2 mg / kg, intravenous) Hartley, male and female, 500-700 g body weight) were used in the experiments of this example. The volume of expansion (mm, mean ± standard deviation) was assessed before and after administration of convulsant (histamine, serotonin, acetylcholine, bradykinin, intravenous) or NaBPC157 (10 minutes prior to repeated convulsive administration). .

result

Swelling volume reduction caused by serotonin, histamine, acetylcholine and bradykinin was inhibited by NaBPC157 (10 μg / kg body weight, intravenous and / or 10 ng / kg body weight, intravenously).

Given the observed beneficial efficacy of NaBPC157 for bronchial contraction caused by various seizure agents and the known close similarity between the model used and the condition of the person, the successful administration of NaBPC157 may lead to the reduction of bronchial contraction and / or respiratory tract. It has been proven to treat various diseases associated with the disorder.

Example 30

Efficacy on Pulmonary Hypertension Syndrome

Materials and Techniques Used

The experiments of this example are intended to demonstrate the efficacy of NaBPC157 and the efficacy of nitric oxide (NO) agonists and antagonists on tissue damage and progression of pulmonary hypertension syndrome (PHS) in chicks. An experiment was conducted to investigate acute toxicity. This experiment included saline (1 mL, intraperitoneal), NaBPC157 (10 μg / kg body weight), L-NAME (NO antagonist / dosage 50, 100, 150 mg / kg body weight) and L-arginine (NO agonist / 100 mg / kg body weight) combinations thereof (L-NAME + NaBPC157, L-NAME + L-arginine) in a single dose. Histopathological and hematological analyzes of the spleen, heart, liver and lung were performed. In addition, chronic toxicity experiments were performed. Animals were taken daily for 5 weeks with L-NAME (10 mg / kg body weight), L-arginine (100 mg / kg body weight), NaBPC157 (10 μg / kg body weight) and combinations thereof (L-NAME + NaBPC157, L-NAME + L-arginine) was administered by intraperitoneal administration. Seven animals were killed weekly from each group, including the control group (saline 1 ml intraperitoneal).

result

PHS was induced by administration of L-NAME in treated chicks. This efficacy is inhibited by simultaneous administration of L-arginine and NaBPC157. Histopathological investigations of both acute and chronic toxicity show that L-NAME causes severe tissue damage (myocardial and hepatic cell necrosis, lymphoid cell necrosis in the spleen), while L-arginine congestion edema and bleeding in all organs. This was noticeably induced. Hematology analysis showed a significant decrease in hemoglobin and leukocyte counts in the L-NAME treated group of chicks. The efficacy of L-NAME was effectively inhibited by the administration of L-arginine and NaBPC157. NaBPC157 did not cause any tissue or organ damage.

Given the similarity between the animal model used above and the disorders of the person concerned, NaBPC157 can be used for the treatment of pulmonary hypertension syndrome. In addition, its administration has proven useful in conventional breeding.

Example 31

Hypertension

Substances used, techniques and results

In hypertensive animals (Goldblatt hypertension) with two kidneys (2K1C) or one kidney (1K1C), NaBPC157 drops blood pressure rapidly (eg within 5 or 10 minutes after injection). This efficacy lasts 20 minutes in 1K1C rats or at least 12 hours in 2K1C animals. This efficacy was then obtained repeatedly after a second or third administration 24 to 48 hours later. Similarly, rats reared on high fructose (80%) or high salt (15%) diets for extended periods of time reduced NaBPC157 levels to near normal levels. This therapeutic effect lasted for a long time and resistance developed even after 6 months. NaBPC157 had no effect on basal blood pressure.

In addition to decreased blood pressure levels, NaBPC157 is also accompanied by severe hyalin degeneration with pronounced parenchymal hemorrhage, arterial wall degeneration, fear of cells in the heart, severe congestion in the adrenal glands (especially the medulla), and hematuria in the kidneys. A severe congestion, parenchymal bleeding and hemangiodeposition and red blood cell phagocytosis in the spleen were greatly alleviated or even eliminated the usual injuries and disorders that are common in various organs (after 3 weeks of increased salt diet in the control). Blood vessels were less damaged in NaBPC157 treated rats in addition to less fear formation and no bleeding in the vessel wall. It was also demonstrated that rats treated with NaBPC157 significantly inhibited the damage to the base.

NaBPC157 administered at 10 μg or 10 ng / kg body weight, intraperitoneal, or gastric, using hypertension from several animal models, yielded robust results with various regimes. Given the apparent similarity between this model and the state of the corresponding human, NaBPC157 can be used for treating hypertension and various organ damages caused by hypertension.

Example 32

Efficacy on Bleeding Time

Materials and Techniques Used

The efficacy of 2-AMP-BPC157 on bleeding time in mice and rats (with and without heparin pretreatment) was investigated in relation to in vitro activity on blood clot formation in human blood.

result

At baseline conditions (2-AMP-BPC157, 10 μg, 10 ng, 10 pg, 1 pg, 10 fg / kg body weight, intraperitoneal, at the same time as tail amputation), reduction of dose dependent bleeding time was observed in both mice and rats. Strongly proven. In the heparin (1000 IU / kg, intraperitoneal) pretreatment experiments (2 hours before initiation of bleeding), it was regularly shown that a significant increase in bleeding time was significantly reduced with 10 μg or 10 ng / kg body weight, intraperitoneally. Efficacy of disease-forming variables in human blood was not demonstrated in in vitro experiments (2-AMP-BPC157, concentration 10 μg / ml, 1 hour incubation). Therefore, it is expected to be effective for the endothelial layer.

Given the close similarity between human and animal bleeding mechanisms and the significance of the animals used for the selected agents, 2-AMP-BPC157 can be used for inhibited coagulation treatment (eg, heparinization). have.

Example 33

Efficacy in Diabetes Testing

Substances used, techniques and results

At certain intraperitoneal administrations (ie, 10 μg / kg body weight), NaBPC157 has been found to not only prevent alloxan progression but also to suppress streptozotocin-induced diabetes in rats. Diabetes was reduced for 14 days and there was less island damage. In our preliminary experiments, NaBPC157 also had efficacy when administered in the presence of 14 days of alloxic damage already formed. In further experiments, the antidiabetic efficacy of NaBPC157 was further studied, focusing on their possible activity when administered intragastrically. NaBPC157 (dose 10 μg and 10 ng / kg body weight) was pretreated (24 hours before or 1 hour prior to dosing aloxane), co-treatment and post-treatment (48 hours after dosing aloxane) at 300 mg / kg subcutaneously or 200 mg / kg body weight, subcutaneously combined with alloxan administration. In general, NaBPC157 reduced the increased glucose serum concentrations and greatly increased the survival rate of alloxane treated animals (efficacy was evident in the group treated with high doses of aloxane). In addition to increased glucose serum concentrations in alloxane treated animals, it is interesting to note that gastrointestinal damage appeared to be worse in rats with diabetes in the control group. Each NaBPC157 regimen significantly reduced the severity of ulcer irrespective of treatment conditions. Similar results were obtained in mice as well.

Given the similarities between human pathology and animals used, in view of a wide range of uses for screening the activity of the formulations, NaBPC157 can be used to treat diabetes.

Example 34

Antitraumatic Water-soluble Effect

Materials and Methods Used

The anti-traumatic efficacy of NaBPC157 was evaluated in comparison with aspirin and morpholine reference materials in various experimental models of indirect / direct traumatic and neurotoxicity, namely writhing (acetic acid / magnesium sulfate), tail tingling, hot plate and capsaicin administration. .

result

NaBPC157 (administered in ng or μg / kg, intraperitoneally) significantly reduced responses in writhing (inflammatory and non-inflammatory, prostaglandin dependent and independent) and tail tingling tests. In the hot plate test, unlike morpholine, NaBPC157 did not show efficacy against normal animals. In addition, NaBPC157 was administered once daily or in pretreated form for 14 days after capsaicin injection. In this test, NaBPC157 significantly reduced capsaicin-pain. This decrease in the efficacy of capsaicin did not appear when NaBPC157 was administered (only 14 days after capsaicin) in the presence of existing capsaicin-bodily neuronal degeneration. Therefore, the consequent somatoneuron depletion after capsaicin administration can be completely eliminated with daily NaBPC157 administration, so the efficacy of NaBPC157 may be associated with the integrity of capsaicin sensitive sensory neurons and their protection [ie, major afferent Neurons include small cell bodies and anhydrous (C-) or thin myelin (A-8) fibers].

Given that capsaicin induces the same inhibitory efficacy in both humans and animals, NaBPC157 can be used not only for the treatment of various pain inhibitions, but also for conditions involving impaired sensory neuronal action.

Example 35

Efficacy in Spasm

Materials and Techniques Used

The efficacy of NaBPC157 on cramps caused by bicuculin, picrotoxin, strykinin and isoniazid was investigated. Convulsives (mg / kg body weight, intraperitoneal). KBPC157 (100 μg, 10 μg or 10 ng / kg body weight 15 minutes before or concurrently with picrotoxin (3), styrikinin (6, 3, or 1.5), bicuculin (2.5) and isoniazid (800 mg) ) Was administered intraperitoneally.

result

KBPC157 always provides positive anticonvulsive efficacy (in terms of dosage and time dependence) for all convulsants administered. Given the similarity between the animal and human condition used, KBPC157 can be used to treat convulsive disease.

Example 36

Spinal cord injury

Materials and Techniques Used

Spinal cord injury was induced in Albino Wistar rat males by adjusting pressure (at blood clip Aesulap 0.10 to 0.15 N) for 30 seconds to the exposed spinal cord (at the level of Th12). Animals were treated immediately with NaBPC157 (10 μg, 10 ng / kg body weight, intraperitoneal) or saline until day 10 after treatment. Animals were killed 24 hours after the last dose.

result

The investigation was performed clinically (evaluated daily, grades 1-5) and using a microscope. Sustained paralysis (control rats) in rats treated with NaBPC157 was found to be significantly alleviated. Given the similarity between the animal and human condition used, NaBPC157 can be used to treat spinal cord injury.

Example 37

Chronic alcoholism

Materials and Techniques Used

The efficacy of NaBPC157 on chronic alcoholism was tested. Male Albino Wistar rats were used for this experiment. Rats were allowed to drink commercial whiskey or ethanol (10-20% aqueous solution) for 3 months. NaBPC157 (10 μg, 10 ng / kg body weight), propranolol (10 mg / kg body weight) and ranitidine (10 mg / kg body weight) were administered by intragastric or intraperitoneal administration (control rats had eastern blood saline 5.0 Administration of ml / kg) as either prophylaxis (10 days before the onset of alcohol) or simultaneous treatment or treatment with alcohol (end of the 2 month period, ie beginning during the last month of the experiment).

result

Direct measurement of blood pressure in the portal vein significantly inhibited and / or decreased the increased portal blood pressure of control rats, as well as propranolol provided in all regimens. In contrast, treatment with linidine showed a greater increase in portal blood pressure than was present in control animals. In addition, beyond propranolol, NaBPC157 can also significantly alleviate the increased damage in the gastrointestinal tract of alcohol treated rats. Similar beneficial effects have been shown for damage in other organs (eg kidney, heart).

Given the generally known fact that increased alcohol intake produces similar changes in humans and this constant beneficial effect, NaBPC157 has proved useful in the treatment of alcohol damage.

Example 38

Efficacy on Cerebral Ischemic Disease

Materials and Techniques Used

NaBPC157 (10 μg or ng / kg body weight, intraperitoneal) or saline (5.0 mL / kg body weight, intraperitoneal) was administered 1 hour before or 1 hour before the rat carotid artery was ligated for 3 or 6 hours.

result

The efficacy of NaBPC157 in both pretreatment (1 hour before ligation) as well as post-treatment (1 hour after ligation) has proven very beneficial. This is the case when used in μg and ng units for all 3 or 6 hour experiments. Apart from the grade, brain tissue edema and severe perivascular edema with cerebral congestion were the most pronounced and characteristic findings. In particular, the cerebellum showed anhydrous areas. All these features were much less in the NaBPC157 treated group of animals regardless of treatment status.

Given the severe ischemic damage seen in the control group and the marked similarity between rat and human ischemic disease, it is clear that NaBPC157 can be used to treat cerebral ischemic disease in humans.

Example 39

Efficacy on Nerve Damage

Materials and Techniques Used

The efficacy of NaBPC157 on peripheral nerve regeneration was examined. In anesthetized Wistar rat adults (225-250 g), the right sciatic nerve was exposed, and the connective tissue was removed and then transversely incised 5 cm apart. Three nerve sheaths (10.0 Ethilon, Ethicon) were used for anastomosis to obtain the proper fiber bundle arrangement. Immediately following anastomosis procedure, the anastomosis site was topically treated with 1 ml of NaBPC157 (2 μg / ml or 2 ng / ml) in a stomach or intraperitoneal (10 μg or 10 ng / kg body weight). Eastern control saline was used as a control. Functional tests were performed on the postoperative marking days (3, 6, 9, 12 and 30 days). These tests include hot water test (60 ° C.), cold water test (20 ° C.), walking track analysis, EMG-spaced latency and CMAP amplitude. Samples for histological and morphological analysis were also taken. In other identical surgical procedures and experiments using the same nerve site, microsurgical forceps were used for 60 seconds to cause left upper injury and topical administration of NaBPC157. Evaluation was performed on days 2, 7, 10, 50 and 100.

result

The healing of injured rats was greatly improved by administering NaBPC157 clinically or using a microscope to evaluate it, particularly in the early periods.

Given the close similarity between animal damage and human condition, NaBPC157 can be used for the treatment of peripheral nerve damage.

Example 40

Efficacy on Neuroleptic Disorders

The efficacy of NaBPC157 on various neuroleptic induced disorders was examined.

Substances used, techniques and results

Administration of NaBPC157 (10 [mu] g or 10 ng / kg body weight, intraperitoneal) was performed with haloperidol and flufenazine [haloperidol 0.625, 1.25, 2.5, 5.0 and 10.0 mg / kg body weight in small amounts of two neuroleptics in subsequent time intervals. , Intraperitoneal, flufenazine 0.3125, 0.625, 1.25, 2.5 and 5.0 mg / kg body weight, intraperitoneal (4.0 ml / kg)]. In addition to the beneficial effects on small amounts of neuroleptics, a much more potent anti-atherogenic effect of NaBPC157 was observed when using NaBPC157 in μg and ng unit doses. In addition, stiffness could not be observed in the control sulfide mice, but this effect was observed in the sensory orientation of animals treated with sulfide (20, 40, 80 and 160 mg / kg body weight, intraperitoneally).

It has proven beneficial to administer NaBPC157 in the treatment of diseases associated with dopamine-based, celiac disease and neuroleptic disorders.

Example 41

에서 의 Administration in Treatment

Materials and Techniques Used

Experimental bleeding shocks were tested with anesthetized rats (common cannula in carotid and jugular veins). Regulated blood volume removal was performed until death or low blood pressure (30-35 mmHg) was stabilized. In this experiment, CaBPC157 (10 μg or 10 ng / kg body weight) or saline was administered 15 minutes prior to bleeding intraperitoneally (5.0 mL / kg) or 5 minutes after low blood pressure stabilization intravenously (3.0 mL / kg).

result

Compared to the values of the control group, a large amount of blood volume loss (dose dependence) persisted before death was observed in CaBPC157 treated rats. In animals with decreased blood volume and hypotension treated with CaBPC157, significant blood pressure increases that persisted rapidly and for long periods of time without death were demonstrated. This contrasts with a 75% mortality rate of mild blood pressure rise in a short time before the end of 45 minutes of the experiment in the control group. In rats treated only postoperatively after anesthesia without vasodilation, CaBPC157 (intraperitoneal or intravenous) administered after 65 minutes of stabilization had no significant effect on arterial blood pressure values within the same postdose period. These data suggest that CaBPC157 is effective in improving the effects of acute blood loss. Given the apparent similarities in animal models used with human disorders, CaBPC157 can be used for the treatment of shock.

Example 42

Efficacy on Abnormal Lymphocytes

Substances used, techniques and results

The immunological findings of two female patients with rare clinical entities--hereditary eosinophilia associated with intraepithelial bullous dermatitis and subacute sclerogenic panencephalitis--were studied. The efficacy of NaBPC157 (in vitro) on lymphocyte chromosome aberration and T cell proliferation was studied. NaBPC157 has been shown to significantly reduce severe types of chromosomal abnormalities (ie, normalized lymphocyte findings) and have a stimulating effect on the mitotic cycle.

Example 43

Efficacy on Malformations

Materials and Techniques Used

The efficacy of NaBPC157 on vitamin A induced malformations in mice was studied. On day 10 of pregnancy NaBPC157 (10 ng / kg body weight or 10 μg / kg body weight, intraperitoneal) or saline (5 ml / kg body weight, intraperitoneal) was simultaneously combined with vitamin A (15,700 IU / kg body weight, intramuscular) Administered. Mice administered with only the same dose of saline at the same time without vitamin A were used as controls.

result

Vitamin A was administered to cause a number of malformations. In the group treated with NaBPC157, the number of malformations was significantly reduced. It has proven effective to administer NaBPC157 in the treatment of fetal disorders.

Example 44

Ovariectomy

Materials and Techniques Used

Normal ovarian resection was performed. NaBPC157 (10 μg or 10 ng / kg body weight, intraperitoneal) was administered once daily for 28 days after ovarian resection or once daily for 14 days from 15 days after ovarian resection. The final dose was 24 hours after the animals were killed in a regular long term regime. The other group received a single dose in μg 15 days after ovarian resection. Five groups were used as controls. Two groups were treated with saline after ablation, and the animals were killed 15 and 28 days after ovarian ablation, one group treated with saline without ablation, and two without ovaries Groups were treated with NaBPC157 (10 μg or 10 ng / kg body weight, intraperitoneally) once daily for 28 days. Vaginal smears were taken from each animal 5 minutes prior to surgery on the 9th, 14th and 28th day of the experiment and stained with Pap for cytological evaluation. Vaginal endothelial maturity was assessed by maturity criteria. Limb bones were collected from each animal for the biomechanical test briefly described below.

Biomechanical testing

All bones were checked for dependence on various moments of bending (one point bending) (bending is moment = Nm) in various directions, forward, backward, and outward. The femur and tibia were used in this experiment. The distal ends of these bones were fixed to a metal tube 1 cm in length with a bone binder (palacose). The metal tube was fixed to the load system. The bone was kept moist. In addition to the length of the free portion of the bone, the length from the fixed point to the load point was measured. The experiment was performed according to the principle of bending a stick fixed to one point within the elastic limit. The loads were 0.1, 0.2, 0.5, 0.7, 1.0 N. The deflection or bending angle was measured as the deflection angle of the laser beam refracted from the small mirror which fixed the end of the bone. The weight of the mirror was added to the applied bending force. A few millimeters of paper irradiated with a laser beam were placed about 1.5-1.8 m away from the bone with the small mirror on which the bone was mounted. The angle of deformation was measured using a triangular technique. During each load, the duration (and recovery) of the strain was measured. After the load was removed, the bone was returned to its starting position, demonstrating that there was no structural change in the bone and that the load was applied within the elastic limits of the hook diagram. These observations indicate that two loads can be applied to the same bone in several directions without deforming the bone structure. If no further deformation was observed during the next two minutes or if additional recovery was obtained, these points were either finalized (generally, the measurement time was about 30 seconds (ovary-reduced control) or 16 minutes (healthy group). )]. Each deformation of the bone (load and post-load recovery) was recorded in mm / sec.

result

Daily administration of two doses of NaBPC157 significantly increased maturity compared to atrophy reduced maturity in rats of untreated controls. Thus, NaBPC157 has been shown to prevent vaginal atrophy in rats induced by castration.

In the control group in which the ovary was excised, the lower the degree of bending for a short time, the lower the degree of recovery. Equally low values were obtained for both controls killed after 15 or 28 days. This was also observed in the ovaries removed and treated with saline (killed on day 15 or 28). On the 15th day after ovarian resection, a positive trend was observed in the group administered with single dose of NaBPC157 in μg. Very good results were obtained in all other groups treated with NaBPC157 in ng or μg. The best results were obtained in the group receiving NaBPC157 daily with 28 μg treatment for 28 days. All variables were significantly improved in the ovarian excised animals and improvements were obtained to obtain the values observed in the healthy group. No differences were found in healthy rats, whether treated with saline or with NaBPC157 (dose in μg or ng units).

Given the significance generally accepted in these animal models for human ovarian dissection for vaginal atrophy and osteoporosis development, it has been demonstrated that NaBPC157 can be used for ovarian resection.

Example 45

tumor

Substances used, techniques and results

One model commonly used as laboratory tumors involves assessing the metastatic number of carcinoma and melanoma B-16 in mice. Like various models of laboratory tumors, these laboratory tumors share significant similarities with the disorders observed in human patients. NaBPC157 was administered in various protocols to reduce the number of metastases in treated mice compared to the control group. Erlich ascites tumor (EAT) is a tumor that can grow in all strains of mice. It can grow in plural or solid form, depending on the manner of administering the tumor cells. Although animal tumor models generally share only a partial similarity with humans, the use of such models is generally acceptable because such models provide a great deal of utility in antitumor drug research.

Survival (days) of mice injected with Erlich ascites tumor cells is limited to less than 25 days in most cases. NaBPC157 (2 μg / ml) and tumor cells were previously incubated to prolong the life of the animals injected with these tumor cells. More than 90% of animals survived to the end of the 45-day observation period. In addition, various cytostatic drugs caused neutropenia in patients as well as experimental animals. Cyclophosphamide is most commonly used as an agent that causes neutropenia. The administration of cyclophosphamide (180 mg / kg, intraperitoneal) causes serious disorders. NaBPC157 inhibits neutropenia, reduces reticulocytes, and increases hemoglobin levels.

In conclusion, NaBPC157 proved to have antitumor activity. Given the pronounced similarity between animal models and human conditions and the beneficial efficacy gained in both in vivo and in vitro experiments, NaBPC157 is useful for antitumor treatment. NaBPC157 is suitable for mitigating cytostatic malignant effects.

Example 46

Antiviral activity

Materials and Techniques Used

ARBO viruses [tick-born encephalitis (TBE), Bhania, Dengue 1, 2, 3, 4, mystery's, West Nile, El Robo), A hepatitis, lymphoid context meningitis (LCM) and Herpes type 1 10 ^- Dilutions of ² (0.02 ml per mouse) were administered intradermal (or post-operative hepatitis A) as a virus suspension. NaBPC157 (20 μg / kg body weight) or 0.9% NaCl (0.02 mL per mouse) intradermal or intraperitoneally a) pretreatment regimen, b) concurrent with virus administration or c) 4 days post infection in the presence of disease symptoms formed Was administered.

result

After simultaneous administration of NaBPC157 with the virus, the onset of signs of disease and a significant delay in mortality were observed (regulated regularly on days 4 and 5 post-infection in animals of the control group not). When administered in the presence of serious disease, prolongation of survival time was observed constantly. This demonstrated no signs of death after NaBPC157 or any subsequent death.

To confirm the results obtained, viral suspensions (ARBO-virus) administered by inoculation with NaBPC157 or saline, respectively, inoculated with brain suspensions obtained from surviving (NaBPC157) (in spite of viral administration) or dead (saline) mice, respectively. The pathogenesis of infected) was tested. Unlike mice inoculated with saline-brain suspension (not different from saline-treated mice inoculated with virus suspension), no signs of disease or death were observed in mice inoculated with NaBPC157-brain suspension. Mice were observed for 50 days after brain suspension inoculation.

NaBPC157 has the beneficial effect of being resistant to incubation at elevated temperatures (56 ° C. for 30 minutes).

Given that such viruses cause similar disorders in humans, NaBPC157 can be used to treat viral diseases, especially in the case of severely impaired overall conditions (e.g. AIDs and AIDS related conditions). have.

Example 47

Bowel damage

Materials and Techniques Used

Experimental model with ulcer [48 hours of constrained stress, subcutaneous cysteamine, gastric 96% ethanol ulcer test, NSAIA injury, DNFB (dinitrofluorobenzene), reflux esophagitis after esophageal end-stem anastomosis procedure (pretreatment, Co-treatment, post-treatment] The efficacy of NaBPC157 (10 μg or 10 ng / kg, intraperitoneal, gastric) in rats was investigated in several rats compared to several reference materials.

result

Only NaBPC157 regimen showed constant efficacy in all test models. Bromocriptine, amatadine, pamotidine, cimetidine and somatostatin are ineffective (bondage stress) or partially efficacious (bromocriptine, DNFB-intestinal damage; sucralate, ranitidine, cholestyramine- Reflux esophagitis). Given the reference peptide, dose dependent protection (cysteamine) and / or partially positive efficacy (relative to treatment status) (ethanol) was obtained using glucagon, NPY and secretin, whereas CCK / 26-30 / Had no effect.

Given that all of these animals are used to select agents commonly used in the treatment of gastrointestinal injuries and that NaBPC157 exhibits certain beneficial effects, it has proven beneficial to administer NaBPC157 in the treatment of total intestinal injuries.

Example 48

Efficacy on Cognitive Diseases

Materials and Techniques Used

Administration of anticholinergic agents (scopolamine, atropine, 10 mg / kg body weight, intraperitoneal) results in significant cognitive deficits in rats and successfully reproduced findings evaluating the behavior of animals in underwater T-shaped mazes Get

result

Compared to the control group, the rats treated with scopolamine and atropine showed a significant increase in error. This cognitive deficit (results corresponding to the values in the control group) is eliminated by simultaneous administration of NaBPC157 (10 μg or 10 ng / kg body weight, intraperitoneal).

Given the significance implied by these models for human cognitive impairment, NaBPC157 has proven useful in the treatment of cognitive diseases.

Example 49

Efficacy in withdrawal disorders

Substances used, techniques and results

NaBPC157 has anticonvulsant effect interacting with GABA system and when administered simultaneously with diazepam (5.0 mg / kg, intraperitoneally, twice daily for 10 days) (10 μg / kg 10 ng / kg, intraperitoneally) ) Diazepam efficacy is improved. NaBPC157 alleviates diazepam resistance and delayed withdrawal efficacy and physical dependence. In the resistance assay, 42 h after conditioning regimen, healthy mice after isoniazid treatment (800 mg / kg, intraperitoneal) when diazepam (5.0 mg / kg, intraperitoneal) were administered to mice pre-conditioned with diazepam alone. Shorter preconvulsion potential was observed than in. This was completely eliminated in animals conditioned with NaBPC157 (2 doses) and diazepam. In physical dependency analysis (analysis at 6, 14, 42 and 72 hours after conditioning treatment), diazepam conditioning than in untreated mice after isoniazid treatment at 52 and 72 hours after conditioning treatment Treated mice showed shorter preconvulsion potential. NaBPC157 (10 μg / kg dose) administered with diazepam delayed this effect until the last time observed. In this group at 6 hour intervals, unlike diazepam conditioning treated mice, the isoniazid preconvulsion potential was much longer than in that control. NaBPC157 did not form any resistance effect.

Given the human condition and the significance of these models, NaBPC157 has proven useful in the treatment of withdrawal disorders.

Example 50

Efficacy on Kidney Disorders

Materials and Techniques Used

Mercury chloride (1 mg / kg, intravenous) or cisplatinum (10 mg / kg, subcutaneously) was administered to cause acute renal failure in experimental animals. If renal insufficiency is induced in this manner, renal insufficiency is closely correlated with the corresponding disorder in a person. Thus, damage induced in rats shares a high degree of similarity with the damage in human patients.

result

NaBPC157 significantly alleviated damage to rats in both pretreated and post-treated states. Unilateral nephrectomy caused significant functional overload and hypertrophy of the remaining kidneys. Diuresis was increased in animals treated with unilateral nephrectomy with NaBPC157 treatment and hypertrophy of the remaining kidneys was decreased. Therefore, in terms of the logic of action of compensatory nephropathy, these findings focus on increasing the function of the remaining kidneys. This is supported by additional biochemical results. These data are also in line with the effects obtained from rats suffering from hypertension who underwent renal artery stenosis and / or unilateral nephrectomy. Administration of NaBPC157 in the treatment of kidney disorders has proven effective.

Example 51

Cellular Immune Responses of Peripheral Blood Lymphocytes

The cellular immune response of peripheral blood lymphocytes to BPC was tested in patients and controls with the various diseases shown in Table 2 below. Peripheral blood T cell responses to BPC are described in deSmet MD et al., "Cellular immune response of patients with uveitis to retinal antigens and their fragments." Am. J. Ophthal., 110: 135-142, 1990] was tested in cell culture for 7 days. Cells were incubated without antigen and with 20 μg / L of antigen (BPC157 pentadecapeptide). For each patient, the stimulation index was calculated by dividing the mean count of the antigen-stimulated cultures by the mean count of the control cell culture without the addition of the antigen, ie BPC157 pentadapeptide. The results are shown in Table 2 below.

The value of the control group ≤ 2 measured in the control subjects corresponds to the values measured by the other investigators for peptides with similar molecular weight, and in the control subjects it was observed that peripheral blood lymphocytes did not induce sensitization to BPC157 pentadecapeptide. . In patients suffering from various diseases, the cellular response to BPC described in Table 2 indicates systemic lymphocyte sensitization to gastric juice peptides. These data indicate that BPC related disorders exist in patients suffering from various diseases. This suggests that BPC-related agents (eg, salts of BPC157 pentadecapeptide) be administered for immunomodulatory treatment of the disorder.

Increased Peripheral Blood T Cell Stimulation Index in 11 Patients with Various Diseases disease gender Stimulus index control value ≤2 Peptic ulcer F 26.82 Multiple sclerosis F 13.80 Subacute Sclerosing Panencephalitis F 2.54 Optic neuritis M 3.54 Optic neuritis F 9.53 Uveitis F 16.87 Uveitis M 15.21 Uveitis F 22.30 Seronegative spondyloarthropathy F 4.99 Hereditary eosinophilia and bullous dermatitis F 2.70 Uveitis F 4.75

The results of the aforementioned pharmacological experiments show that the activity of BPC peptide salts protects the organism from stress and disease and is generally beneficial in normalizing organ function. In addition, the peptide salts of the present invention have efficacy in the prevention and treatment of various diseases and conditions in humans and / or animals.

Claims (15)

BPC (body protective compound) peptide salt comprising 8 to 15 amino acids, the anion of which is a negatively charged peptide having the formula (I), the cation of which is a cation of an inorganic or organic nontoxic base, a BPC peptide salt . Formula I [Zaa Pro Pro Pro Xaa Yaa Pro Ala] ^{(-) or (2-)} In formula (I), Xaa is a neutral aliphatic amino acid residue, Yaa is a basic amino acid residue, Zaa is an acidic amino acid residue. 2. The cation of claim 1, wherein the cation of the salt is an alkali or alkaline earth metal, Zn ²⁺ , primary amine, secondary amine and tertiary amine, in particular Na ⁺ , K ⁺ , Li ⁺ , Cs ⁺ , Ca ²⁺ , NH _B ⁺ peptide salt selected from the group consisting of ₄ ⁺ , triethanolamine ⁺ , cyclohexylamine ⁺ , 2-AMP ⁺ (2-amino-1-propanol) or TRIS [tris- (hydroxymethyl) aminomethane]. The method according to claim 1 or 2, wherein Xaa is Ala, bAla, Leu, Ile, Gly, Val, Nle or Nva, Yaa is Lys, Arg, Orn or His, Zaa is Glu, Asp, Aad or Apm BPC peptide salt. The BPC peptide salt according to any one of claims 1 to 3, further comprising a pharmaceutically or diagnostically acceptable carrier. The BPC peptide salt according to any one of claims 1 to 4, further comprising trehalose. The compound according to any one of claims 1 to 5, wherein the chemical formula is BPC peptide salt. The method according to any one of claims 1 to 6, wherein the peptide is And BPC peptide salt selected from the group consisting of. 8. The BPC peptide salt according to claim 1, wherein the peptide is cyclized by an amide bond, in particular between the first and last amino acid residues. 9. The BPC peptide salt according to any one of claims 1 to 8, wherein the salt is dissolved in an aqueous solution or an aqueous / alcoholic solution, preferably at a pH of 6.0-8.5. A pharmaceutical composition having a compatible, storage stability comprising a pharmaceutically effective amount of a BPC peptide salt according to any one of claims 1 to 9 and optionally a physiologically acceptable carrier. The pharmaceutical composition of claim 10 for oral use in admixture with trehalose. 12. A diagnostic, storage stable composition comprising a diagnostically effective amount of a BPC peptide salt according to any one of claims 1-11. Disorders related to nitric oxide (NO) formation or impaired NO-system function, in particular hypertension, angina, erectile dysfunction, circulatory system, sepsis, seizures, infection, respiratory disorder syndrome, adhesion and aggregation of platelets and leukocytes, endothelial dysfunction Gastrointestinal damage, peristaltic disorders, diabetes, pancreatitis, hypotension and Parkinson's disease; Dysfunction or hyperfunction of the sensory sensory nerves, especially sensory neuropathy, herpes neuralgia, atopic dermatitis, healing of damaged tissues, urticaria, psoriasis, bullous stool, eczema, photodermatitis, acquired cold or high temperature Chronic arthritis, gastrointestinal damage, specific or nonspecific reactive hyperactivity of the upper or lower airways (asthma, rhinitis); Endothelial disorders; Wounds, ulcers; Conditions associated with acute infection and / or chronic infection, in particular chronic arthritis, diseases associated with delayed type hypersensitivity, and gastrointestinal damage; Liver disease, especially organ damage caused by free radicals specifically caused by light irradiation; Diseases associated with catecholaminergic disorders, in particular schizophrenia, amphetamine administration effects, drug abuse; Stress related symptoms; Acute pancreatitis, especially acute pancreatitis with concomitant gastroduodenal pathology; Heart disorders; Congestive disorders; Parkinson's disease and Parkinson's disease-like pathology; Temperature dependence disorder; Bone damage; Damage to various organs due to hypertension; Coagulation disorders; Pain disorders; Convulsive disorders; Spinal cord injury; Alcoholic damage caused by alcohol abuse or increased alcohol intake; Cerebral ischemic disease; Peripheral nerve damage; Celiac disease and neuroleptic disorders; Diseases associated with abnormal or mutated lymphocytes; Fetal disorders; Osteoporosis and vaginal atrophy due to ovarian resection procedure; tumor; Viral diseases, in particular AIDS or ARC; Gastrointestinal damage; Cognitive disease; Withdrawal disorders; Use of the BPC peptide salt according to any one of claims 1 to 9 for the manufacture of a pharmaceutical composition having a suitability according to claim 10, storage stability for the treatment of kidney disorders and cellular immune response disorders. Use of the BPC peptide salt according to any one of claims 1 to 9 for the preparation of a pharmaceutical composition having compatibility, storage stability according to claim 10 as a cell protective and organ adjuvant. Mixing at least one BPC peptide with at least one base in an aqueous solvent or an aqueous / alcoholic solvent to obtain a BPC peptide salt, wherein the cation of the salt is a cation of an inorganic or organic non-toxic base; A method for preparing a BPC peptide salt according to any one of claims 9 to 9.